Salivary and serum interleukin-6 levels in proliferative verrucous leukoplakia
Cytokines and chemokines have been analysed in patients with oral squamous cell carcinoma and potentially malignant disorders. We selected interleukin-6 (IL-6) because it is a multifunctional interleukin reported to be altered in potentially malignant oral disorders and in malignant lesions. To date, this has not been evaluated or tested in proliferative verrucous leukoplakia (PVL), however.
This study aimed to analyse the differences in serum and saliva IL-6 levels among patients with PVL, oral squamous cell carcinoma (OSCC) and healthy controls and to examine the relationship between salivary IL-6 levels and the extent of the verrucous area.
Using an enzyme-linked immunosorbent assay, we determined the serum and saliva IL-6 levels in three groups: 20 patients with PVL, 20 with OSCC and 20 healthy controls.
There were significant (p < 0.01) differences in the serum and saliva IL-6 levels among the three groups and among the three grades of extent of the verrucous areas (p = 0.01). In the OSCC group, there was a significant difference in the saliva IL-6 levels between patients with and without lymph node metastasis at diagnosis (p = 0.02).
We found that patients with OSCC had the highest salivary and serum IL-6 levels, while PVL had lower values than OSCC, but higher than the controls, and these altered levels were associated with the extent of the verrucous areas.
Salivary and plasma IL-6 are altered in patients with PVL, with more extensive verrucous areas being associated to the highest IL-6 levels. This could be a significant tool for monitoring patients with PVL, their progression to more advances stages and their recurrences.
KeywordsProliferative verrucous leukoplakia Interleukin-6
Hansen et al.  first described proliferative verrucous leukoplakia (PVL) in 1995. This is a potentially malignant disorder with a risk of malignant transformation into oral squamous cell carcinoma (OSCC) of up to 70 %, according to some authors [2, 3, 4, 5, 6, 7].
The aetiology of PVL is unknown, although human papillomavirus (HPV) has been implicated [8, 9, 10]. Others were not able to detect any definitive relationship with HPV [11, 12]. Kresty et al.  described some genetic changes in these patients, and Kkanrit et al.  reported a ploidy anomaly. Tobacco does not play a significant role in PVL [2, 5, 15].
PVL starts as simple white homogenous leukoplakia but progresses to verrucous forms, affecting many different oral locations . There are only few large case series reported [1, 2, 3, 5]. In 2012, we published a series of 55 patients [36 (65.5 %) females, 19 (34.5 %) males; mean age 61.69 ± 11.76 years] . The PVL patients who develop oral cancers were more commonly female and non-smokers. Those who developed more than one OSCC were most likely to develop gingival PVL lesions.
Criteria for the diagnosis of proliferative verrucous leukoplakia 
1. Leukoplakia showing the presence of verrucous or wart-like areas, involving more than two oral subsites. The following oral subsites are recognised: dorsum of the tongue (unilateral or bilateral), border of the tongue, cheek mucosa, alveolar mucosa or gingiva upper jaw, alveolar mucosa or gingiva lower jaw, hard and soft palate, floor of the mouth, upper lip and lower lip
2. When adding all involved sites, the minimum seize should be at least 3 cm
3. A well-documented period of disease evolution of at least 5 years, being characterised by spreading and enlarging and the occurrence of one or more recurrences in a previously treated area
4. The availability of at least one biopsy in order to rule out the presence of a verrucous carcinoma or squamous cell carcinoma
Cytokines and chemokines have been analysed in patients with OSCC [19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29], and in potentially malignant disorders, most commonly interleukin-6 (IL-6) [19, 21, 22, 30, 31, 32, 33]. IL-6 is a multifunctional cytokine produced by macrophages, neutrophils, fibroblasts, keratinocytes and endothelial cells . It can be anti- and proinflammatory , has some characteristics of a growth factor and is involved in the development and progression of some cancers [36, 37, 38].
However, no study has examined these cytokines in PVL. Therefore, we compared the serum and saliva IL-6 levels among patients with PVL and OSCC and healthy controls and observed whether there was a relationship between the IL-6 level and the extent of the verrucous lesions in PVL.
Materials and methods
The ethics committee of Valencia University approved this study (reference H1391893856722). Our sample consisted of three groups of patients: 20 with PVL, 20 with OSCC and 20 healthy controls.
The diagnosis of PVL was established following the criteria of van der Waal et al.  (Table 1). Patients with a malignancy were excluded from the PVL group. In patients with PVL, we also determined the number of oral areas affected, considering the tongue, buccal mucosa, alveolar mucosa, gingiva, hard and soft palate, floor of the mouth and lips . We distinguished three grades of the extent of the verrucous areas in different oral locations: grade 1, the verrucous areas affected were ≤33 % of the oral mucosa; grade 2, the area affected was between 34 and 66 %; and grade 3, the lesions affected were ≥67 % of the oral mucosa.
Diagnosis of OSCC was made from a biopsy of the oral lesion. The control group was selected from healthy people who were not taking any medication and had no lesions or inflammatory processes in their oral cavity. There was no difference in age among the three groups (p < 0.05).
In every case, a blood sample was obtained from a peripheral arm vein before starting any treatment after a diagnosis was made. A sample of non-stimulated saliva was obtained following the method of Navazesh et al. . The saliva and blood were centrifuged immediately at 3000 rpm for 15 or 10 min, respectively. Then, the samples were frozen at −80 °C until the IL-6 was determined. To prevent the contamination of saliva samples with blood, we recommended the participants not to brush their teeth for 45 min before sample collection. Saliva samples visibly contaminated with blood were discarded, and new saliva samples were obtained. Blood and saliva samples were collected at a second visit, 1 week after the first.
The IL-6 in the samples was assayed using a commercial enzyme-linked immunosorbent assay (ELISA; Quantikine Human IL-6; R&D Systems, Minneapolis, MN, USA). Briefly, saliva and plasma samples were loaded on microplates pre-coated with IL-6-specific monoclonal antibody. Standards and samples are bound by the immobilised antibody. After washing to remove unbound material, an enzyme-linked polyclonal antibody specific for IL-6 was added to the wells. After a second wash with buffer, the substrate solution was added and incubated for 30 min and then protected from light. The reactions were stopped with the appropriate substrate. The optical density of the colour in each well was measured at 450 nm. IL-6 levels are reported as means ± SD, expressed in picograms per millilitre .
The Kruskal–Wallis test was used to compare the means of the blood and saliva levels among the three groups. The Mann–Whitney U test was used to compare the two patient groups. The relationship between IL-6 in saliva and that in plasma was analysed using Spearman’s rank correlation coefficient. Statistical significance was accepted at p < 0.05.
Clinical characteristics of the PVL and OSCC patients
PVL (group 1)
OSCC (group 2)
5 (25 %) males, 15 (75 %) females
15 (75 %) males, 5 (25 %) females
Tongue (6, 30 %)
Tongue (9, 45 %)
Buccal mucosa (17, 85 %)
Buccal mucosa (2, 10 %)
Gingiva (20, 100 %)
Gingiva (3, 15 %)
Palate (13, 65 %)
Palate (1, 5 %)
Floor of the mouth (13, 65 %)
Floor of the mouth (3, 15 %)
Lip (5, 25 %)
Retromolar area (2, 10 %)
Number of locations per patient
2 (4 cases, 20 %)
3 (4 cases, 20 %)
4 (2 cases, 10 %)
5 (6 cases, 30 %)
6 (4 cases, 20 %)
Grade 1 (11 cases, 55 %)
Grade 2 (6 cases, 30 %)
Grade 3 (3 cases, 15 %)
White plaque lesions with verrucous areas and, in some cases, erythroplastic lesions
Ulceration (12 cases, 60 %)
Tumour (3 cases, 15 %)
Mixed (5 cases, 25 %)
Size of the lesion
Lymph node Metastasis
Positive (6 cases, 30 %)
Serum and saliva IL-6 levels in the three groups
Serum IL-6 (mean ± SD) (pg/mL)
Saliva IL-6 (mean ± SD) (pg/mL)
133.34 ± 46.52
Kruskal–Wallis test = 29.33
p < 0.01
151.59 ± 129.27
Kruskal–Wallis test = 40.64
p < 0.01
199.49 ± 214.70
435.04 ± 142.15
21.27 ± 5.63
33.40 ± 38.95
Differences in the saliva and serum IL-6 levels according to the clinical characteristics of the groups
Saliva IL-6 (mean ± SD) (pg/mL)
Serum IL-6 (mean ± SD) (pg/mL)
Group 1: PVL (20 cases)
Males (5 cases)
125.96 ± 83.08
Mann–Whitney U = 37
p > 0.05
114.10 ± 36.76
Mann–Whitney U = 20
p > 0.05
Females (15 cases)
160.13 ± 142.81
139.76 ± 48.73
Grade of verrucous leukoplakia
Grade 1 (11 cases)
71.97 ± 37.50
Kruskal–Wallis test = 8.9
p = 0.01
142.82 ± 56.85
Kruskal–Wallis test = 1.3
p > 0.05
Grade 2 (6 cases)
200.85 ± 131.33
112.80 ± 18.29
Grade 3 (3 cases)
345.01 ± 101.67
139.69 ± 41.71
Group 2: OSSC (20 cases)
Males (15 cases)
437.69 ± 152.29
Mann–Whitney U = 32
p > 0.05
210.31 ± 238.30
Mann–Whitney U = 31
p > 0.05
Females (5 cases)
427.07 ± 121.27
167.02 ± 135.81
Lymph node metastasis
Negative (14 cases)
392.48 ± 150.93
Mann–Whitney U = 14
p = 0.02
215.31 ± 251.60
Mann–Whitney U = 41.5
p > 0.05
Negative (6 cases)
534.34 ± 25.48
162.57 ± 90.80
Type of the lesion
Ulcerative (13 cases)
446.03 ± 95.96
Kruskal–Wallis test = 0.46
p > 0.05
149.76 ± 87.24
Kruskal–Wallis test = 1.6
p > 0.05
Tumour (2 cases)
524.10 ± 37.58
137.35 ± 18.42
Mixed (5 cases)
370.83 ± 241.85
353.63 ± 395.45
In the OSCC group, there were no differences in the saliva and serum IL-6 levels with age or the type of lesion (p > 0.05). There was a significant difference in the saliva IL-6 of patients with lymph node metastasis compared to those without metastasis at diagnosis (p = 0.02). There was no correlation between the size of the lesions and the saliva (Spearman’s ρ = −0.69, p > 0.05) or serum (Spearman’s ρ = 0.25, p > 0.05) levels of IL-6.
Cytokines are proteins that participate in many normal and pathological processes that affect cells, including their growth, differentiation and death . Some cytokines have proinflammatory actions, including interferon-gamma, tumour necrosis factor alpha (TNF-α) and IL-1β . In contrast, transforming growth factor beta 1 (TGF-β1)  serves an inflammatory function.
Interleukin-6 is a multifunctional cytokine that participates in the immune response and inflammation and boosts the growth of malignant cells [37, 42]. Nibali et al.  reported that IL-6 levels change in association with several dental problems including periodontitis , dental granulomas, periapical lesions , oral lichen planus , recurrent aphthous stomatitis  and Sjögren’s syndrome .
Some authors have detected increased levels of different saliva interleukins, such as TNF-α [19, 30, 33], IL-6 [19, 21, 30, 33], IL-8 , IL-1β  and IL-1α , in potentially malignant disorders. In addition, the levels of TNF-α , IL-6 [21, 22, 31, 32], IL-8  and IL-1β  are increased in the serum of patients with potentially malignant disorders.
In OSCC, the saliva interleukins TNF-α [19, 24, 30], IL-6 [19, 20, 21, 23, 24, 29, 30], IL-8 [20, 24, 26, 28, 30], IL-1β , IL-1α [24, 30] and vascular endothelial growth factor (VEGF)  are elevated. Changes in TNF-α , IL-6 [22, 25, 26], IL-1β  and VEGF  levels in OSCC have also been described. As seen above, the most commonly reported altered IL is IL-6.
In premalignant and malignant lesions, IL-6 levels have been described to be increased in relation to the controls . IL-6 seems to contribute to oral cancer pathogenesis through different mechanisms . IL-6 enhances matrix metalloproteinases 1 and 9  and plays a role in cell growth and differentiation of keratinocytes . It has been described that IL-6 inactivates the p53 tumour suppression gene . As a result, IL-6 can promote tumour cell proliferation . These mechanisms could explain the higher IL-6 levels in PVL compared to the controls, being higher in cases where cancer has already developed. We also found higher levels of IL-6 in PVL cases with more extensive verrucous areas. Therefore, the usefulness and clinical relevance of this finding, which has not being described to date, is that we could identify progression in patients with PVL based on the determination of IL-6 in plasma and saliva.
Although several authors have reported changes in IL-6 of the serum and saliva in patients with potentially malignant disorders, to the best of our knowledge, this is the first study to report significantly (p < 0.05) higher saliva and serum IL-6 levels in patients with PVL compared to a control group. However, these levels were lower in PVL than in our OSCC patients.
IL-6 promotes the growth of epithelial cells [37, 38] and plays an important role in infiltrative growth and in the development of metastasis . Reflecting this, we found a significantly higher IL-6 level in the saliva of OSCC patients with, than in those without, lymph node metastasis. Sato et al.  found no relationship among tumour size, gender and salivary IL-6 levels, which we also noted. They also did not find a correlation between the salivary IL-6 concentrations and gender, duration of disease, T stage, tobacco use or periodontal disease.
There is a correlation between salivary IL-6 levels and early locoregional recurrence in patients with OSCC . Performing a multivariate analysis after surgery, Sato et al.  found that the salivary IL-6 concentration was an independent risk factor for locoregional recurrence (p = 0.03; risk ratio 0.14). These findings are in concordance with the increased salivary IL-6 levels in our OSCC patients with lymph node metastasis.
PVL is potentially a highly malignant disorder. Currently, the disease is diagnosed clinically and histologically. Recently, van der Waal’s group combined these to propose diagnostic criteria . It would be useful to find a laboratory test that is correlated with the aggressiveness and extent of the verrucous areas in the oral cavity.
Saliva is very easy to collect and might be a useful complementary test material for determining the diagnosis of PVL. In this regard, we found that both salivary and serum IL-6 were elevated compared to controls (p < 0.05), with salivary levels better able to distinguish PVL from controls (Table 2). In addition, when we analysed the grades of the verrucous area in the oral cavity, there was a significant (p < 0.05) difference, with the highest salivary IL-6 being correlated with the greatest verrucous extent (Table 4).
The aetiology of PVL is not known , and the disease shows a very high rate of malignant transformation into oral squamous cell carcinoma . It is a high-risk potentially malignant disorder. PVL starts as simple homogeneous leukoplakia, but in time, changes to multiple verrucous leukoplastic areas. There are non-laboratory tests which could predict the progression of PVL to its most advance stages, before oral squamous cell carcinoma develops. The usefulness and clinical relevance of our finding that IL-6 levels are higher in cases with more extensive verrucous areas is that it could help us to identify progression in these patients. This has not been previously described.
Shkeir et al.  have already reported that IL-6 could be of predictive value for head and neck squamous cell carcinoma (HNSCC) progression, because they recorded higher IL-6 levels in patients with metastasis, as we have also found. Duffy et al.  also proposed that pretreatment serum IL-6 analysis could be a valuable biomarker for predicting recurrences and overall survival in HNSCC. The relationship between the size of the metastasis and IL-6 levels has not been described to date.
It would be interesting to analyse the relationship between the size of metastasis and the IL-6 levels in the context of a new prospective trial focusing on oral cancer only. However, our study centres mainly on PVL, where IL-6 had not been evaluated to date.
Our finding could also be used to monitor the treatment of these PVL cases, because recurrences usually manifest with new verrucous areas. Therefore, if salivary and plasma IL-6 are higher in more extensive verrucous areas, then the IL-6 levels can be expected to be higher in recurrences. However, in order to firmly accept this statement it would be advisable to conduct a new prospective study with a larger number of cases in which IL-6 is evaluated before treatment and over follow-up with recurrences.
In conclusion, patients with OSCC had the highest salivary and serum IL-6 levels. PVL had lower values than OSCC, but higher than the controls, and these altered levels were associated with the extent of the verrucous area.
Conflict of interest
The authors declare that they have no competing interests.
- 10.Gopalakrishnan R, Weghorst CM, Lehman TA, Calvert RJ, Bijur G, Sabourin CL, Mallery SR, Schuller DE, Stoner GD (1997) Mutated and wild-type p53 expression and HPV integration in proliferative verrucous leukoplakia and oral squamous cell carcinoma. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 83:471–477CrossRefPubMedGoogle Scholar
- 19.Jureti M, Cerovi R, Belui-Gobi M, Brekalo Pro I, Kqiku L, Palj S, Pezelj-Ribari S (2013) Salivary levels of TNF- and IL-6 in patients with oral premalignant and malignant lesions. Folia Biol 59:99–102Google Scholar
- 21.Brailo V, Vucicevic-Boras V, Lukac J, Biocina-Lukenda D, Zilic-Alajbeg I, Milenovic A, Balija M (2012) Salivary and serum interleukin 1 beta, interleukin 6 and tumor necrosis factor alpha in patients with leukoplakia and oral cancer. Med Oral Patol Oral Cir Bucal 17:e10–e15CrossRefPubMedPubMedCentralGoogle Scholar
- 25.Vairaktaris E, Yapijakis C, Serefoglou Z, Avgoustidis D, Critselis E, Spyridonidou S, Vylliotis A, Derka S, Vassiliou S, Nkenke E, Patsouris E (2008) Gene expression polymorphisms of interleukins-1 beta, -4, -6, -8, -10, and tumor necrosis factors-alpha, -beta: regression analysis of their effect upon oral squamous cell carcinoma. J Cancer Res Clin Oncol 134:821–832CrossRefPubMedGoogle Scholar
- 26.St John MA, Li Y, Zhou X, Denny P, Ho CM, Montemagno C, Shi W, Qi F, Wu B, Sinha U, Jordan R, Wolinsky L, Park NH, Liu H, Abemayor E, Wong DT (2004) Interleukin 6 and interleukin 8 as potential biomarkers for oral cavity and oropharyngeal squamous cell carcinoma. Arch Otolaryngol Head Neck Surg 130:929–935CrossRefPubMedGoogle Scholar
- 27.Chang KP, Chang YT, Liao CT, Yen TC, Chen IH, Chang YL, Liu YL, Chang YS, Yu JS, Wu CC (2011) Prognostic cytokine markers in peripheral blood for oral cavity squamous cell carcinoma identified by multiplexed immunobead-based profiling. Clin Chim Acta 412:-12Google Scholar
- 38.Prasad G, McCullough M (2013) Chemokines and cytokines as salivary biomarkers for the early diagnosis of oral cancer. Int J Dent. 2013: Article 813756, 7 pagesGoogle Scholar
- 41.Shkeir O, Athanassiou-Papaefthymiou M, Lapadatescu M, Papagerakis P, Czerwinski MJ, Bradford CR, Carey TE, Prince ME, Wolf GT, Papagerakis S (2013) In vitro cytokine release profile: predictive value for metastatic potential in head and neck squamous cell carcinomas. Head Neck 35(11):1542–1550CrossRefPubMedPubMedCentralGoogle Scholar
- 44.Prso IB, Kocjan W, Simi H, Brumini G, Pezelj-Ribari S, Borci J, Ferreri S, Karlovi IM (2007) Tumor necrosis factor-alpha and interleukin 6 in human periapical lesions. Mediators Inflamm. 2007:38210Google Scholar